Biotinylated isocoumarins

ABSTRACT

Biotinylated isocoumarins, their use in inhibiting serine proteases with chymotrypsin-like and elastase-like specificity and in purifying proteins.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a novel class of heterocyclic compounds usefulfor selectively inhibiting chymotrypsin-like enzymes, selectivelyinhibiting elastase or for generally inhibiting serine proteases of manyclasses. We have found that isocoumarins substituted with biotin andhydrophobic groups are potent inhibitors of chymases, elastases, andother serine proteases. These biotinylated isocoumarins can be used toselectively or generally remove proteases from solution or biologicalsystems. They can be used either selectively or generally to isolateserine proteases. They can also be used to selectively or generallyinhibit serine proteases either in vitro or in biological systems.

2. Description of the Related Art

Serine proteases play critical roles in several physiological processessuch as digestion, blood coagulation, complement activation,fibrinolysis, viral infection, fertilization, and reproduction. Serineproteases are not only a physiological necessity, but also a potentialhazard if they are not controlled. Uncontrolled proteolysis by elastasesmay cause pancreatitis, emphysema, rheumatoid arthritis, bronchialinflammation and adult respiratory distress syndrome. Humanpolymorphonuclear leukocyte elastase may also be involved in blistering.Accordingly, specific and selective inhibitors of these proteases shouldbe potent anti-inflammatory agents useful in the treatment ofprotease-related diseases (Powers and Harper, in Proteinase Inhibitors,Barrett and Salvesen, eds., Elsevier, 1986, pp 55-152, incorporatedherein by reference). In vitro proteolysis by chymotrypsin or theelastase family is a serious problem in the production, purification,isolation, transport or storage of peptides and proteins. It is oftendesirable to remove all proteases from solution or biological systems.In other cases, it is desirable to isolate or remove a specific serineprotease.

Anti-inflammatory agents are used to treat elastases-associatedinflammation including rheumatoid arthritis and emphysema. Although thenaturally occurring protease inhibitor, α1-protease inhibitor (α1-PI)has been used to treat patients with emphysema, this inhibitor is notwidely used clinically due to the high dosage needed for the treatmentand difficulty of producing large quantities. Therefore small molecularweight elastase inhibitors are needed for therapy.

SUMMARY OF THE INVENTION

It is an object of this invention to find a novel group of specificbiotinylated inhibitors for elastase, chymotrypsin and other serineproteases of similar substrate specificity and for serine proteases ingeneral. Inhibitors are compounds that reduce or eliminate the catalyticactivity of the enzyme. Trypsin and trypsin-like enzymes normally cleavepeptide bonds in proteins and peptides where the amino acid residue onthe carbonyl side of the split bond (P₁ residue) is Lys or Arg. Elastaseand elastase-like enzymes, on the other hand, cleave peptide bonds wherethe P₁ amino acid is Ala, Val, Ser, Leu and other similar amino acids.Chymotrypsin and chymotrypsin-like enzymes hydrolyze peptide bonds whereP₁ amino acid is Trp, Tyr, Phe, Met, Leu or other amino acid residueswhich contain aromatic or large alkyl side chains. All of the aboveenzymes have extensive secondary specificity and recognize amino acidresidues removed from the P₁ residue.

It is an object of this invention to discover new biotinylated proteaseinhibitors, especially elastase inhibitors, and chymase inhibitors.These inhibitors are useful for controlling tissue damage and variousinflammatory conditions mediated by proteases particularly elastases.The inhibitors of this invention would also be useful for controllinghormone processing by serine proteases and for treating diseases relatedto tryptases and chymases such as inflammation and skin blistering.

It is a further object of this invention to find a novel group ofspecific biotinylated inhibitors useful in vitro for inhibiting trypsin,elastase, chymotrypsin and other serine proteases of similar specificityand for inhibiting serine proteases in general. Such inhibitors could beused to identify and isolate new proteolytic enzymes encountered inresearch. They could also be used in research and industrially toprevent undesired proteolysis that occurs during the production,isolation, purification, transport and storage of valuable peptides andproteins. Such proteolysis often destroys or alters the activity and/orfunction of the peptides and proteins. Uses would include the additionof the inhibitors to antibodies, enzymes, plasma proteins, tissueextracts or other proteins and peptides which are widely sold for use inclinical analyses, biomedical research, and for many other reasons. Forsome uses a specific inhibitor would be desirable, while in other cases,an inhibitor with general specificity would be preferred. Thebiotinylated inhibitors could also be used to remove proteases, isolateserine proteases from solution or biological systems, or to detectproteases in solution or in biological systems by utilizing the affinityor biotin for avidin either on columns or in solution. Numerous avidinderivatives containing enzymes, heavy metal markers, antigens, orantibodies are commercially available which could be use to react withserine protease containing a biotinylate inhibitory moiety covalentlyattached to the protease.

DETAILED DESCRIPTION OF THE INVENTION

Isocoumarins substituted with hydrophobic groups have been found to beexcellent inhibitors of several serine proteases including humanleukocyte elastase, porcine pancreatic elastase, bovine chymotrypsin andhuman leukocyte cathespsin G. These compounds inhibit the serineproteases by reaction with the active site serine to form an acylenzyme, which in some cases may further react with another active sitenucleophile to form an additional covalent bond. These structures may beused in vivo to treat diseases such as emphysema, adult respiratorydistress syndrome, rheumatoid arthritis and pancreatitis which resultfrom uncontrolled proteolysis by elastase, chymotrypsin, trypsin andrelated serine proteases. These inhibitors may be used in vitro toprevent proteolysis which occurs in the process of production,isolation, purification, storage or transport of peptides and proteins.The inhibitors also contain a biotin moiety which can be recognized byavidin and avidin conjugates. The novel substituted isocoumarin andrelated heterocyclic compounds have the following structural formula:##STR1## or a pharmaceutically acceptable salt, wherein

Z is selected from the group consisting of H, halogen, C₁₋₆ alkyl, C₁₋₆fluorinated alkyl, C₁₋₆ alkyl substituted with R¹, C₁₋₆ fluorinatedalkyl substituted with R¹, C₁₋₆ alkoxy, C₁₋₆ fluorinated alkoxy, C₁₋₆alkoxy substituted with R¹, C₁₋₆ fluorinated alkoxy substituted with R¹,C₁₋₆ alkyl with a phenyl group attached to the alkyl group, C₁₋₆ alkoxywith a phenyl group attached to the alkoxy group, C₁₋₆ alkyl with anattached phenyl group substituted with R², C₁₋₆ alkyl with an attachedphenyl group disubstituted with R², C₁₋₆ alkoxy with an attached phenylgroup substituted with R², C₁₋₆ alkoxy with an attached phenyl groupdisubstituted with R²,

wherein R² represents halogen, COOH, OH, CN, NO₂, NH₂, C₁₋₆ alkyl, C₁₋₆alkoxy, C₁₋₆ alkylamine, C₁₋₆ dialkylamine, C₁₋₆ alkyl-O--CO--, C₁₋₆alkyl-O--CO--NH--, or C₁₋₆ alkyl-S--,

wherein R¹ represents halogen, COOH, OH, CN, NO₂, NH₂, C₁₋₆ alkoxy, C₁₋₆alkylamine, C₁₋₆ dialkylamine, C₁₋₆ alkyl-O--CO--, or C₁₋₆alkyl-O--CO--NH--, C₁₋₆ alkyl-S--, or tosylamino,

wherein Spacer is any organic structure which is 0-25 Å long,

wherein T represents --NH--, --O--, or --S--, and

Y is selected from the group consisting of H, halogen, trifluoromethyl,methyl, OH and methoxy.

Alternately the novel isocoumarin and related heterocyclic compound arerepresented by structure (I) where,

Z is selected from the group consisting of H, halogen, C₁₋₆ alkyl, C₁₋₆fluorinated alkyl, C₁₋₆ alkyl substituted with R¹, C₁₋₆ fluorinatedalkyl substituted with r¹, C₁₋₆ alkoxy, C₁₋₆ fluorinated alkoxy, C₁₋₆alkoxy substituted with R¹, C₁₋₆ fluorinated alkoxy substituted with R¹,C₁₋₆ alkyl with a phenyl group attached to the alkyl group, C₁₋₆ alkoxywith a phenyl group attached to the alkoxy group, C₁₋₆ alkyl with anattached phenyl group substituted with R², C₁₋₆ alkyl with an attachedphenyl group disubstituted with R², C₁₋₆ alkoxy with an attached phenylgroup substituted with R², C₁₋₆ alkoxy with an attached phenyl groupdisubstituted with R²,

wherein R² represents halogen, COOH, OH, CN, NO₂, NH₂, C₁₋₆ alkyl, C₁₋₆alkoxy, C₁₋₆ alkylamine, C₁₋₆ dialkylamine, C₁₋₆ alkyl-O--CO--, C₁₋₆alkyl-O--CO--NH--, or C₁₋₆ alkyl-S--,

wherein R¹ represents halogen, COOH, OH, CN, NO₂, NH₂, C₁₋₆ alkoxy, C₁₋₆alkylamine, C₁₋₆ dialkylamine, C₁₋₆ alkyl-O--CO--, or C₁₋₆alkyl-O--CO--NH--, C₁₋₆ alkyl-S--, or tosylamino,

wherein Spacer represents --[NH--(CH₂)_(n) --CO]_(n) --,--[NH--(CH₂)_(n) --NH--CO]_(n) --, --[NH--C₆ H₄ --CO]_(n) --, --[NH--C₆H₄ --NH--CO]_(n) --, --NH--(CH₂)_(n) --CO--NH--(CH₂)_(n) --NH--CO--,--NH--(CH₂)_(n) --CO--NH--(CH₂)₃ --NH--(CH₂)₃ --NH--CO--CH₂ CH₂ --CO--,or --(AA)_(n) --, where n=1-6,

wherein AA is a side chain blocked or unblocked amino acid with the Lconfiguration, D configuration, or no chirality at the α-carbon selectedfrom the group consisting of alanine, valine, leucine, isoleucine,proline, methionine, methionine sulfoxide, phenylalanine, tryptophan,glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine,aspartic acid, glutamic acid, lysine, arginine, histidine,phenylglycine, beta-alanine, norleucine, norvaline, alpha-aminobutyricacid, epsilonaminocaproic acid, citrulline, hydroxyproline, ornithine,homoarginine, sarcosine, indoline 2-carboxylic acid,2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylicacdi), O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine,S-benzylcysteine, NH₂ --CH(CH₂ CHt₂)--COOH, alpha-aminoheptanoic acid,NH₂ --CH(CH₂ -1-naphthyl)-COOH, NH₂ --CH(CH₂ -2-naphthyl)-COOH, NH₂--CH(CH₂ -cyclohexyl)-COOH, NH₂ --CH(CH₂ -cyclopentyl)-COOH, NH₂--CH(CH₂ -cyclobutyl)-COOH, NH₂ --CH(CH₂ -cyclopropyl)-COOH,trifluoroleucine, or hexafluoroleucine,

wherein T represents --NH--, --O--, or --S--, and

Y is selected from the group consisting of H, halogen, trifluoromethyl,methyl, OH and methoxy.

The compounds of Formula (I) can also contain one or more substituentsat position B as shown in the following structure: ##STR2##

wherein electronegative substituents such as NO₂, Cn, Cl,COOR, and COOHwill increase the reactivity of the isocoumarin, and electropositivesubstituents such as NH₂, OH, alkoxy, thioalkyl, alkyl, alkylamino, anddialkylamino will increase its stability. Neutral substituents couldalso increase the stability of acyl enzyme and improve the effectivenessof the inhibitors.

The complete structure of biotin in the biotin-Spacer moiety is shownbelow. ##STR3##

Other substituted isocoumarins have been prepared earlier for otherpurposes (illustrative examples: 3-chloroisocoumarin, Davies and Poole,J. Chem. Soc., pp 1616-1629(1928); 3-chloro and 3,4-dichloroisocoumarin,Milevskaya, Belinskaya, and Yagupol'skii, Zhur. Org. Khim. 9, pp2145-2149(1973); 3-methyl and 4-carboxy-3-methylisocoumarin, Tirodkarand Usgaonkar, Ind. J. Chem. 7, pp 1114-1116(1969); 7-nitro and7-aminoisocoumarin, Choksey and Usgaonkar, Ind. J. Chem. 14B, pp 596-598(1976), the preceding articles are incorporated herein by reference).

A number of other substituted isocoumarins have been prepared recentlyfor inhibition of serine proteases (3-chloroisocoumarin, Harper, Hemmi,and Powers, J. Am. Chem. Soc. 105, pp 6518-6520(1983);3,4-dichloroisocoumarin, Harper, Hemmi, and Powers, Biochemistry 24, pp1831-1841 (1985); 3-alkoxy-7-amino-4-chloroisocoumarin, Harper andPowers, J. Am. Chem. Soc 106, pp 7618-7619(1984), Harper and Powers,Biochemistry 24, 7200-7213 (1983); substituted isocoumarins with basicgroups (aminoalkoxy, guanidino or isothiureidoalkoxy), Kam, Fujikawa andPowers, Biochemistry 27, pp 2547-2557 (1988); 7-substituted3-alkoxy-4-chloroisocoumarins, Powers, Kam, Narasimhan, Oleksyszyn,Hernandez and Ueda, J. Cell Biochem. 39, pp 33-46 (1989), Powers,Oleksyszyn, Narasimhan, Kam, Radhakrishnan and Meyer, Jr. Biochemistry29, 3108-3118 (1990), the preceding articles are incorporated herein byreference; Powers and Harper, U.S. Pat. No. 4,596,822; Powers and Kam,U.S. Pat. No. 4,845,242; Powers, Kam, Oleksyszyn, Glinski, andHernandez, U.S. Pat. No. 5,089,633; Powers and Kam, U.S. Pat. No.5,089,634 which are also incorporated by reference).

The following compounds are representative of the invention:

7-biotinylamino-4-chloro-3-propyloxyisocoumarin

7-biotinylamino-4-chloro-3-(2-phenylethoxy)isocoumarin

7-(6-biotinylaminocaproyl)amino-4-chloro-3-ethoxyisocoumarin

7-(6-biotinylaminocaproyl)amino-4-chloro-3-propyloxyisocoumarin

7-(6-biotinylaminocaproyl)amino-4-chloro-3-(2-phenylethoxy)isocoumarin

7-[6-(6-biotinylaminocaproyl)aminocaproyl]amino-4-chloro-3-(2-phenylethoxy)isocoumarin

7-[6-(6-biotinylaminocaproyl)aminocaproyl]amino-4-chloro-3-methoxyisocoumarin

7-biotin-NH(CH₂)₂ NH--COCH₂NHCONH-4-chloro-3-(2-phenylethoxy)isocoumarin

It has been found that compounds of Formula (I) are effective inhibitorsof the proteolytic function of human leukocyte elastase as shown inTable I. Compounds of Formula (I) are also effective inhibitors of theproteolytic function of chymotrypsin and pancreatic elastase as shown inTable I. Compounds of Formula (I) are also effective in the preventionof unnecessary proteolysis caused by chymotrypsin and elastase in theprocess of purification, transport and storage of peptides and proteinsas shown in Table I by effective inhibition of chymotrypsin andelastase.

Compounds of Formula (I) with a group consisting of biotinylamino withbiotinylamino group attached to alkanoylamino, Y group of Cl, and Zgroup of phenylethoxy group are effective in the inhibition of ratgranule chymase as shown in Table II. The reactivation of inhibited PPE,chymotrypsin, or rat granule chymase by these biotin isocoumarins in thepresence of hydroxylamine as shown in Table III, and IV is useful in thepurification of these enzymes from enzyme mixtures or granules byapplying the inhibited granules to the avidin beads, where thebiotinylated enzymes form tight complex with avidin and are retained onthe column. Finally the enzyme can be reactivated and eluted off thecolumn with a hydroxylamine solution.

The tight complex of biotin-avidin has been used as a powerful tool forpurifying proteins. One such example is Williams et al., J. Biol. Chem.264, pp 7536-7545 (1989). A biotinylated-ε-aminocaproyl-peptidechloromethylketone was used to react with an active protease to form thebiotinylated inactivated enzyme which was retained on the avidin beads.This procedure allows removal of the protease from enzyme and zymogenmixture. In contrast to the Williams procedure, the use of biotinylatedisocoumarins allows either an inactivated serine protease to be formedor a reactivatable serine protease to be formed depending on the natureof the bond linking the spacer to the isocoumarin (changing this bondfrom an amide to a urea linkage changes the ratio of irreversiblealkylation to reversible acylation). Thus, we have the option ofobtaining active enzyme from the avidin column. This is not possiblewith the Williams procedure which uses an irreversible chloromethylketone inhibitor.

Although little information is available on the structure of biotinbinding site of avidin, the spacer between the biotin and the ligandmolecule such as the isocoumarin, chloromethyl ketone or insulin iscrucial for the binding of a biotinylated ligand to avidin. Green et al.(Biochem. J. 125, pp 781-791 (1971)) attempted to determine the depth ofthe biotin binding site on avidin by studying the effect of chain lengthof ω-bis(biotinyldiamines) on avidin polymer formation. They concludedthat stable polymers were formed when the chain linking the carboxylgroups of the biotins was 18 Å long and the carboxyl group must lieabout 8-9 Å beneath the surface of the avidin molecule. Finn et al.(Biochemistry 23, pp 2554-2558 (1984)) also calculated that the distancebetween the carboxyl group of dethiobiotin and the N-terminal aminogroup of the insulin B-chain would be 9.77 Å fordethiobiotinyl-A1-insulin, 18.36 Å for dethiobiotinyl-A2-insulin, and25.52 Å for dethiobiotinyl-A1-DPA-insulin (A1, A2, and A1-DPA haddifferent chain length of spacers). Thus, any of these ligands shouldhave sufficient space between the dethiobiotinyl and insulin portions tobind normally to avidin. However, only the longest of the three ligandsshowed the same rate of dissociation from Suc-avidin as dethiobiotinitself. Therefore, spacer arms are required for optimizing theinteraction between the biotinylated ligand and the avidin complex, andlonger spacers are preferred.

The biotin-avidin interaction is very useful in many areas such asimmunoassays, receptor studies, immunocytochemical staining and proteinisolation. In the enzyme immunoassay system, the biotinylated antibodyis bound to the immobilized antigen or primary antibody, and avidin canbe conjugated with enzymes, fluorochromes, ferritin or colloidalmarkers. The biotin-avidin interaction can also be used in blottingtechniques for detecting proteins. It is very useful in the staining ofcellular antigenic determinants. A wide variety of biotinylated primaryprobes such as monoclonal antibodies, lectins, vitamins, sugars,hormones and lipoproteins have been used. This specific interaction hasalso been used successfully in the selective retrieval of labelledplasma membrane components (Orr, J. Biol. Chem. 256, pp 761-766 (1981)).Biotinylated proteins can be used as probes of protein structure andprotein-protein interaction (Billingsley et al. Biotechniques 5, pp22-31 (1987)).

Inactivation rates of serine proteases by substituted isocoumarins weremeasured by the incubation method. An aliquot of inhibitor (25 or 50 μl)in Me₂ SO was added to a buffered enzyme solution (0.01-2.3 μM) toinitiate the inactivation. Aliquots (50-100 μl) were withdrawn atvarious intervals and the residual enzymatic activity was measured. Me₂SO concentration in the reaction mixture was 8-12% (v/v). 0.1M Hepes,0.5M NaCl, pH 7.5 buffer was utilized for the assays of chymotrypsin,cathepsin G (cat G), porcine pancreatic elastase (PPE), human leukocyteelastase (HLE) and rat granule serine proteases. 0.1M Hepes, 0.01MCaCl₂, pH 7.5 buffer was utilized for the assays of trypsin and humanrecombinant (HR) granzyme A. The inhibitor concentrations are shown inthe Tables I and II. Peptide nitroanilides with appropriate sequencewere used as substrates for various serine proteases. All peptide4-nitroanilide hydrolysis was measured at 410 nm (ε₄₁₀ =8800M⁻¹ cm⁻¹ ;Erlanger et al., Arch. Biochem. Biophys. 95, pp 271-278 (1961),incorporated herein by reference). Peptide thioester hydrolysis rateswere measured with assay mixtures containing 4,4'-dithiodipyridine (ε₃₂₄=19800M⁻¹ cm⁻¹ ; Grasetti & Murray, Arch. Biochem. Biophys. 119, pp.41-49 (1967), incorporated herein by reference). First orderinactivation rate constant (k_(obs)) were obtained from plots of ln(v_(t) /v_(o)) vs time, and the correlation coefficients were greaterthan 0.98.

Table I shows the inactivation rate constants of chymotrypsin, cathepsinG (cat G), porcine pancreatic elastase (PPE), human leukocyte elastase(HLE), trypsin and human recombinant (HR) granzyme A inhibited bybiotinylated isocoumarins. The compound with biotin-spacer-T group of6-biotinylaminocaproylamino, Y group of Cl and Z group of phenylethoxyis a good inhibitor for chymotrysin. The structures with biotin-Spacer-Tgroup of 6-biotinylaminocaproylamino, Y group of Cl and Z group ofpropoxy, ethoxy or methoxy are the best inhibitors for HLE. Thesebiotinylated isocoumarins which do not contain a positive charged groupare poor inhibitors of trypsin and HR granzyme (a trypsin-like enzyme).

Table II shows the inhibition of rat granule chymase and tryptase bybiotinylated isocoumarin derivatives. The structure with biotin-spacer-Tgroup of 6-biotinylaminocaproylamino, Y group of Cl and Z group ofphenylethoxy inactivated chymase instantly 50%, and also inhibitedtryptase very slowly. Table III shows the reactivation of inhibited PPE,chymotrypsin, trypsin and rat granule chymase by biotinylatedisocoumarins in buffer and in the presence of hydroxylamine. Inhibitedchymotrypsin regained 10-15% of enzyme activity in the buffer after twodays, but regained 100% of activity in the presence of hydroxylamine.Inhibited rat granule chymase regained 30-100% of activity in thepresence of hydroxylamine. Table IV shows the effect of avidin andstreptavidin on reactivation of inhibited PPE and chymotrypsin. Avidindoes not show any effect, but streptavidin enchanced reactivation ofinhibited chymotrypsin and PPE about 10-20%.

Pulmonary emphysema is a disease characterized by progressive loss oflung elasticity due to the destruction of lung elastin and alveoli. Thedestructive changes of lung parentchyma associated with pulmonaryemphysema are caused by uncontrolled proteolysis in lung tissues(Janoff, Chest 83 pp 54-58 (1983)). A number of proteases has been shownto induce emphysema in animals (Marco et al., Am. Rev. Respir. Dis. 104,pp 595-598 (1971); Kaplan, J. Lab. Clin. Med. 82, pp 349-356 (1973)),particularly human leukocyte elastase (Janoff, ibid 115, pp 461-478(1977)). Leukocyte elastase and other mediators of inflammation alsoappear to play a role in diseases such as mucocutaneous lymph nodesyndrome (Reiger et al., Eur. J. Pediatr. 140, pp 92-97 (1983) and adultrespiratory distress syndrome (Stockley, Clinical Science 64, pp 119-126(1983); Lee et al., N. Eng. J. Med. 304, pp 192-196 (1981); Rinaldo,ibid 301, 900-909 (1982 )).

It is known that in vitro activity of elastase inhibitors correlateswith in vivo activity in animal models of emphysema and inflammation(Otterness et al., editor, Advances in Inflammation Research, Vol. 11,Raven Press 1986, and this article is incorporated herein by reference).Prophylactic administration of an inhibitor of elastase significantlydiminishes the extent of elastase-induced emphysema (Kleinerman et al.,Am. Rev. Resir. Dis. 121, pp 381-387 (1980); Lucey et al., Eur. Respir.J. 2, pp 421-427 (1989)). Thus the novel inhibitors described hereshould be useful for the treatment of emphysema and inflammation.Elastase inhibitors have been used orally, by injection or byinstillation in the lungs in animal studies (Powers, Am. Rev. Respir.Dis., 127, s54-s58 (1983); Powers and Bengali, Am. Rev. Respir. Dis.134, pp 1097-1100 (1986) and these two articles are incorporated hereinby reference). The inhibitors described above can be used by any ofthese routes.

For treatment of inflammation, the compounds of Formula (I) may beadministered orally, topically or parenterally. The term parenteral asused includes subcutaneous injection, intravenous, intramuscular,intrasternal injection or infusion techniques. The dosage dependsprimarily on the specific formulation and on the object of the therapyor prophylaxis. The amount of the individual doses as well as theadministration is best determined by individually assessing theparticular case.

The pharmaceutical compositions containing the active ingredient may bein a form suitable for oral use, for example as tablets, troches,lozenges, aqueous or oily suspensions, dispersible powders or granules,emulsions, hard or soft capsules or syrups or elixirs. Dosage levels ofthe order to 0.2 mg to 140 mg per kilogram of body weight per day areuseful in the treatment of above-indicated conditions (10 mg to 7 gmsper patient per day). The amount of active ingredient that may becombined with carrier materials to produce a single dosage form willvary depending upon the host treated and the particular mode ofadministration.

For injection, the therapeutic amount of the compounds of Formula (I) ortheir pharmaceutically acceptable salts will normally be in the dosagerange from 0.2 to 140 mg/kg of body weight. Administration is made byintravenous, intramuscular or subscutaneous injection. Accordingly,pharmaceutical compositions for parenteral administration will containin a single dosage form about 10 mg to 7 gms of compounds of Formula (I)per dose. In addition to the active ingredient, these pharmaceuticalcompositions will usually contain a buffer, e.g. a phosphate bufferwhich keeps the pH in the range from 3.5 to 7 and also sodium chloride,mannitol or sorbitol for adjusting the isotonic pressure.

A composition for topical application can be formulated as an aqueoussolution, lotion, jelly or an oily solution or suspention. A compositionin the form of an aqueous solution is obtained by dissolving thecompounds of Formula (I) in aqueous buffer solution of pH 4 to 6.5 andif desired, adding a polymeric binder. An oily formulation for topicalapplication is obtained by suspending the compounds of Formula (I) in anoil, optionally with the addition of a swelling agent such as aluminiumstearate and/or a surfactant.

To use the above inhibitors in vitro, they are dissolved in an organicsolvent such as dimethylsulfoxide or ethanol, and are added to anaqueous solution containing serine proteases. The final concentration ofthe organic solvent should be less than 25%. The inhibitors may also beadded as solids or in suspension. The serine protease inhibitors of thisinvention would be useful in a variety of experimental procedures whereproteolysis is a significant problem. Inclusion of these inhibitors in aradioimmunoassay experiments would result in higher sensitivity. The useof these inhibitors in plasma fractionation procedures would result inhigher yields of valuable plasma proteins and would make purification ofthe proteins easier. The inhibitors disclosed here could be used incloning experiments utilizing bacterial cultures, yeast and purifiedcloned product in higher yield.

The following examples are given to illustrate the invention and are notintended to limit it in any manner.

EXAMPLE 1 Preparation of7-(biotinylamino)-4-chloro-3-(2-phenylethoxy)isocoumarin

Biotin acid chloride was prepared by incubating 0.4 g (1.6 mmole) ofbiotin in 6 ml of thionyl chloride at 25°-35° C. for 1 hr, and excessthionyl chloride was removed under vacuum. The acid chloride was usedfor the next step without further purification. Biotin acid chloride and7-amino-4-chloro-3-(2-phenylethoxy)isocoumarin (0.26 g, 0.8 mmole) wasdissolved in small amount of DMF, and then Et₃ N (0.08 g, 0.08 mmole)were added. The reaction mixture was stirred at r. t. overnight. Theproduct was purified by column chromatography, yield 34%, mp 182°-185°C.; TLC, R_(f) =0.25 (CH₂ Cl₂ :MeOH=15:1), NMR was consistent with theassigned structure, mass spectra (FAB⁺) m/e=542 (M+1). Anal. Calc forC₂₇ H₂₈ N₃ O₅ CIS 0.25 H₂ O: C, 59.39; H, 5.22, N, 7.70. Found: C,59.08; H, 5.37; N, 7.94.

7-(Biotinylamino)-4-chloro-3-(pentafluoropropoxy)isocoumarin can beprepared by the same procedure.

EXAMPLE 2 Preparation of7-[(6-biotinylamino)caproyl]amino-4-chloro-3-(2-phenylethoxy)isocoumarin

6-(Biotinylamino)caproic acid was prepared from N-hydroxysuccinimidobiotinate (Jasiewicz et al., Exp. Cell Res. 100, pp 213-217 (1976)) and6-aminocaproic acid methyl ester hydrochloride by a previously describedmethod (Hofmann et al., Biochemistry 23, pp 2547-2553 (1984)).6-(Biotinylamino)caproic acid chloride was prepared by incubating6-(biotinylamino)caproic acid (0.36 g, 1 mmole) in 4 ml of thionylchloride at 25°-35° C. for 1 h, and excess thionyl chloride was removedunder reduced pressure. The residue was dissolved in DMF and7-amino-4-chloro-3-(2-phenylethoxy)isocoumarin (0.34 g, 1 mmole) and Et₃N (0.1 g, 1 mmole) were added and the reaction mixture was stirred atr.t. overnight. The product was purified from column chromatography andeluted with CH₂ Cl₂ :MeOH=10:1, yield, 28%, mp 163°-167° C., NMR wasconsistent with the assigned structure, mass spectra (FAB⁺) m/e=655(M+1). Anal. Calc. for C₃₃ H₃₉ N₄ O₆ ClS.H₂ O: C, 58.87; H, 6.14; N,8.32; Cl, 5.27. Found: C, 58.72; H, 6.22; N, 8.90; Cl, 5.50.

7-[(6-Biotinylamino)caproyl]amino-4-chloro-3-(pentafluoropropoxy)isocoumarincan be prepared by the same procedure.

EXAMPLE 3 Preparation of 7-biotinylamino-4-chloro-3-propyloxyisocoumarin

This compound was prepared similarly as7-biotinylamino-4-chloro-3-(2-phenylethoxy)isocoumarin from biotin acidchloride and 7-amino-4-chloro-3-propyloxyisocoumarin, yield 20%, mp127°-131° C., NMR was consistent with the assigned structure, massspectra (FAB⁺) m/e=502 (M+Na+1). Anal. Calc. for C₂₂ H₂₆ N₃ O₅ ClS.H₂ O:C, 53.06; H, 5.67; N, 8.44; Cl, 7.12. Found: C, 53.30; H, 5.67; N, 8.49;Cl, 7.03.

EXAMPLE 4 Preparation of7-[(6-biotinylamino)caproyl]amino-4-chloro-3-propyloxyisocoumarin

This compound was prepared similarly as7-[(6-biotinylamino)caproyl]amino-4-chloro-3-(2-phenylethoxy)isocoumarinfrom 6-(biotinylamino)caproic acid chloride and7-amino-4-chloro-3-propyloxyisocoumarin, yield 25%, mp 141-145° C., NMRwas consistent with the assigned structure, mass spectra (FAB⁺) m/e=593(M+1). Anal. Calc. for C₂₈ H₃₇ N₄ O₆ ClS.0.5H₂ O: C, 55.14; H, 6.13; N,9.53; Cl, 6.04. Found: C, 54.81; H, 6.26; N, 9.47; Cl, 5.93.

EXAMPLE 5

Preparation of7-[(6-biotinylamino)caproyl]amino-4-chloro-3-ethoxyisocoumarin.

This compound was prepared similarly as7-[(6-biotinylamino)caproyl]amino-4-chloro-3-(2-phenylethoxy)isocoumarinfrom 6-(biotinylamino)caproic acid chloride and7-amino-4-chloro-3-ethoxyisocoumarin, yield 15%, mp 156°-162° C., NMRwas consistent with the assigned structure, mass spectra (FAB⁺) m/e=601(M+Na+1). Anal. Calc. for C₂₇ H₃₅ N₄ O₆ ClS.H₂ O: C, 55.03; H, 6.43; N,9.17; Cl, 5.80. Found: C, 54.81; H, 6.26; N, 9.23; Cl, 5.69.

EXAMPLE 6 Preparation of7-[6-(6-biotinylaminocaproyl)aminocaproyl]amino-4-chloro-3-(2-phenylethoxy)isocoumarin.

6-(6-Biotinylaminocaproyl)aminocaproic acid was prepared from thereaction of 6-biotinylaminocaproic acid and 1,1'-carbonyldiimidazole(CDI) with 6-aminocaproic acid methyl ester, subsequent alkalinehydrolysis of the corresponding methyl ester, and acidification.6-(6-Biotinylaminocaproyl)aminocaproic acid (0.5 g, 1.1 mmole) wasdissolved in 15 ml of DMF at 70° C. and cooled to 40° C., then7-amino-4-chloro-3-(2-phenylethoxy)isocoumarin (0.4 g, 1.3 mmole) wasadded, followed by the addition of 1-hydroxybenzotriazole (HOBt, 0.172g, 1.3 mmole) and diisopropylcarbodiimide (0.16 g, 1.3 mmole). Themixtures were stirred at r.t. overnight, and the solvent was evaporated.The crude product was purified on a silica gel column which was elutedwith CHCl₃ :MeOH:HOAc=65:10:3. The eluted product contained HOBt whichwas then removed by washing with 1N HCl several times. The final productwas obtained as a yellow solid, yield 33%, mp 163°-165° C., NMR wasconsistent with the assigned structure, mass spectra (FAB⁺) m/e=768(M+1). Anal. Calc. for C₃₉ H₅₀ N₅ O₇ ClS: C, 60.96; H, 6.56; N, 9.11;Cl, 4.60. Found: C, 60.72; H, 6.6; N, 9.04; Cl, 4.67.

EXAMPLE 7 Preparation of7-[6-(6-biotinylaminocaproyl)aminocaproyl]amino-4-chloro-3-methoxyisocoumarin.

6-(6-Biotinylaminocaproyl)aminocaproic acid was prepared by the sameprocedure as described in Example 6.6-(6-Biotinylaminocaproyl)aminocaproic acid (0.65 g, 1.4 mmole) wasdissolved in 15 ml of DMF at 70° C. and cooled to 40° C., then7-amino-4-chloro-3-methoxyisocoumarin (0.37 g, 1.7 mmole) was added,followed by the addition of HOBt (0.22 g, 1.7 mmole) and1,3-dicyclohexylcarbodiimide (DCC, 0.34 g, 1.7 mmole). The mixtures werestirred at r. t. overnight, and the solvent was evaporated. The crudeproduct was purified on a silica gel column which was eluted with CHCl₃:MeOH:HOAc=65:10:2. The eluted product contained HOBt which was removedby washing with 1N HCl several times. The final product was identifiedby NMR and mass spectra (FAB⁺) m/e=677 (M⁺).

EXAMPLE 8 Preparation of 7-biotin-NH-(CH₂)₂ NH-COCH₂NHCONH-4-chloro-3-(2-phenylethoxy)isocoumarin.

Biotinylethylenediamine.HCl. Biotin (1 g, 4.1 mmole) was dissolved in 20ml of DMF at 70° C. and cooled to 40° C., 1,1'-carbonyl diimidazole(0.97 g, 6 mmole) in 3 ml of DMF was then added and white precipitateswere appeared. After stirring at r. t. for two hours, ethylenediamine(1.34 ml, 20 mmole) in 10 ml of DMF was added and stirred for another 3hours. After DMF was evaporated, the semisolid residue was dissolved in50 ml of refluxed methanol and the unreacted biotin was removed byfiltration. After the solution was evaporated to dryness, the residuewas washed with CHCl₃ to remove the imidazole, dissolved in 6 ml ofwater, acidified to pH 3.0 with 1N HCl, and evaporated to dryness. Theproduct was crystallized from methanol to give 1.04 g (yield 79%), TLC:R_(f) =0.21 (butanol:acetic acid:H₂ O=4:1:1), mp 241°-242° C., NMR isconsistent with the structure.

Biotin-NH(CH₂)₂ NH--COCH₂ NH.HCl. Biotin-NH(CH₂)₂ NH--COCH₂ NH-Boc wasprepared from the reaction of biotinylethylenediamine.HCl, DCC andt-butyloxycarbonylglycine (Boc-Gly) in the presence of triethylamine inDMF, yield 53%, TLC: R_(f) =0.55 (butanol:acetic acid:H₂ O=4:1:1), mp136°-139° C., NMR was consistent with the assigned structure, massspectra (FAB⁺) m/e=444.5 (M+1). Deblocking the Boc group frombiotin-NH(CH₂)₂ NH--COCH₂ NH-Boc with trifuoroacetic acid at 0° C. andaddition of saturated HCl in ethyl acetate to the residue gave theproduct, yield 81%, TLC: R_(f) =0.32 (butanol:acetic acid:H₂ O=4:1:1),mp 146°-148° C., NMR was consistent with the assigned structure.

7-Biotin-NH--(CH₂)₂ NH--COCH₂NHCONH-4-chloro-3-(2-phenylethoxy)isocoumarin was synthesized from thereaction of 7-amino-4-chloro-3-(2-phenylethoxy)isocoumarin andbiotin-NH(CH₂)₂ NH--COCH₂ NH.HCl. After the isocoumarin (0.43 g, 1.35mmole) and CDI (0.24 g, 1.48 mmole) were dissolved in 8 ml of DMF at 0°C. and stirred at r.t. for 4 h, the solution of biotinylated derivative(0.47 g, 1.23 mmole) and triethylamine (0.17 ml, 1.23 mmole) in DMF wasadded. The reaction mixture was stirred at r.t. for 24 h, thendecolorized, filtered, and evaporated to give dark greenish residue. Theresidue was washed with water and 0.5N HCl, and then applied on a silicagel column which was eluted with CHCl₃ :MeOH (5:1). The final productwas obtained as yellowish green solid, yield 13%, TLC: R_(f) =0.46(CHCl₃ :MeOH=5:1), mp 192°-193° C. (dec), NMR was consistent with theassigned structure, mass spectra (FAB⁺) m/e=686 (M+1). Anal. Calc. forC₃₂ H₃₇ N₆ O₇ ClS: C, 56.09; H, 5.44; N, 12.27. Found: C, 55.92; H,5.46; N, 12.15.

EXAMPLE 9 Elution of inhibited PPE by7-[6-(6-biotinylaminocaproyl)aminocaproyl]amino-4-chloro-3-methoxyisocoumarinfrom avidin-agarose column with NH₂ OH.

PPE (1 mg, 0.04 mmole) was inhibited by biotinylated isocoumarin (0.5mmole) in 0.1M Hepes, 0.5M NaCl, pH 7.5 buffer at 25° C. for 10 min, 7%of residual enzyme activity was found. Excess of inhibitors was removedby Sephadex G-25 column and the inhibited PPE was eluted with 0.1Macetate, 0.5M NaCl, pH 5.0 buffer and had 16% of enzyme activity. Thisinhibited enzyme solution was then applied on the avidin-agarose columnand 34% of protein went through (measured by optical density at 280 nm).The column was washed with pH 5.0 acetate buffer, then eluted with 0.1MHepes, 0.5M NaCl, and 0.5M NH₂ OH, pH 7.5 buffer and 37% of protein waseluted out (measured by OD₂₈₀) from the column. In these proteinfractions, 40% of PPE activity was detected.

                                      TABLE I                                     __________________________________________________________________________    Inhibition of Serine Proteases by Biotin-Isocoumarin Derivatives.sup.a.                            k.sub.obs /[I] (M.sup.-1 s.sup.-1)                       Compounds            Chymotrypsin.sup.b                                                                    Cat. G.sup.c                                                                       HLE.sup.d                                                                         PPE.sup.e                                                                         Trypsin.sup.f                                                                      HR Granyzme                    __________________________________________________________________________                                                   A.sup.g                        7-biotinylamino-4-chloro-                                                                          330     NI.sup.h                                                                           740 NI  1.1                                 3-(2-phenylethoxy)isocoumarin (1)                                                                  165                                                      7-biotinylamino-4-chloro-                                                                           65     6.7  19,900                                                                            470 2.2                                 3-propoxyisocoumarin (2)                                                      7-(6-biotinylaminocaproyl)amino-4-chloro-                                                          1,080   13%.sup.i                                                                          670 NI  1.0  NI                             3-(2-phenylethoxy)isocoumarin (3)                                                                  190                                                      7-(6-biotinylaminocaproyl)amino-                                                                   260     3.3  76,700                                                                            350 10.6 NI                             4-chloro-3-propoxyisocoumarin (4)                                             7-(6-biotinylaminocaproyl)amino-                                                                   260     59   96,000                                                                            520 16.6 26%.sup.i                      4-chloro-3-ethoxyisocoumarin (5)                                              7-[6-(6-biotinylaminocaproyl)aminocaproyl]-                                                                2.2  37,500        5%.sup.i                      amino-4-chloro-3-methoxyisocoumarin (6)                                       7-[6-(6-biotinylaminocaproyl)aminocaproyl]-                                                                NI   230          NI                             amino-4-chloro-3-(2-phenylethoxy)-                                            isocoumarin (7)                                                               7-biotin-NH(CH.sub.2).sub.2 NH--COCH.sub.2 NHCONH-                                                 640     19%.sup.i                                                                          6.6          18%.sup.i                      4-chloro-3-(2-phenylethoxy)isocoumarin (8)                                                         100                                                      __________________________________________________________________________     .sup.a Inhibition was measured in 0.1 M Hepes, 0.5 M NaCl (or 0.01 M          CaCl.sub.2), pH 7.5 buffer, 5-10% Me.sub.2 SO and at 25° C.            SucVal-Pro-NA (0.48 mM) was used as the substrate for chymotrypsin and ca     G. MeOSuc-Ala-Ala-Pro-Val-NA (0.24-0.47 mM) and SucAla-Ala-Ala-NA             (0.29-0.48 mM) were used as the substrate for HLE and PPE respectively.       ZGlu-Phe-Arg-NA (0.032 mM) was a substrate for trypsin. ZArg-SBzl (0.120      mM) was a substrate for human recombinant (HR) granzyme A.                    .sup.b Inhibitor concentrations were 20-400 μM.                            .sup.c Inhibitor concentrations were 75-400 μM.                            .sup.d Inhibitor concentrations were 1.6-210 μM.                           .sup.e Inhibitor concentrations were 38-78 μM.                             .sup.f Inhibitor concentrations were 41-42 μM.                             .sup.g Inhibitor concentrations were 160-210 μM.                           .sup.h No inhibition after 10 min of incubation of inhibitor and enzyme.      .sup.i Percentage of inhibition after 10 min of incubation of inhibitor       and enzyme.                                                              

                                      TABLE II                                    __________________________________________________________________________    Inhibition of Rat granule Serine Proteases by Biotin-Isocoumarin              Derivatives.sup.a.                                                                                [I] Rat Granule Chymase                                                                      Rat Granule Tryptase                       Compounds           (mM)                                                                              % of inhibition.sup.b                                                                    k.sub.obs /[I] (M.sup.-1 s.sup.-1)         __________________________________________________________________________    7-(6-Biotinylaminocaproyl)amino-4-chloro-                                                         0.078                                                                             30-50       6-12                                      3-(2-phenylethoxy)isocoumarin                                                 7-Biotinylamino-4-chloro-                                                                         0.2 10-20      2-3                                        3-(2-phenylethoxy)isocoumarin                                                 __________________________________________________________________________     .sup.a Inhibition was measured in 0.1 M Hepes, 0.5 M NaCl, pH 7.5 buffer,     10% Me.sub.2 SO and at 25° C. SucPhe-Leu-Phe-SBzl (0.14 mM)            ZGly-Arg-SBzl (0.06 mM) were used to measure chymase and tryptase activit     respectively.                                                                 .sup.b Inhibition was not time dependent.                                

                                      TABLE III                                   __________________________________________________________________________    Reactivation of Inhibited PPE, Chymotrypsin and Rat Granule Chymase           by Biotin-Isocoumarins in Buffer and in the Presence of                       NH.sub.2 OH.sup.a.                                                                              % of Enzyme Activity Reactivated                                                                         Rat granule                                        PPE        Chymotrypsin                                                                             Trypsin                                                                            chymase                          Inhibitor         in buffer.sup.b                                                                    +NH.sub.2 OH                                                                        in buffer.sup.b                                                                    +NH.sub.2 OH                                                                        in buffer.sup.b                                                                    +NH.sub.2 OH                     __________________________________________________________________________    7-biotinylamino-4-chloro-3-(2-phenyl-                                                                      15%  100%       100%                             ethoxyisocoumarin (1)                                                         7-(6-biotinylaminocaproyl)amino-                                                                           10%  100%       30-50%                           4-chloro-3-(2-phenylethoxy)-                                                  isocoumarin (3)                                                               7-biotinylamino-4-chloro-3-propoxy-                                                             35%  98%                                                    isocoumarin (2)                                                               7-(6-biotinylaminocaproyl)amino-                                                                37%  90%              33%                                   4-chloro-3-propoxyisocoumarin (4)                                             7-(6-biotinylaminocaproyl)amino-                                                                66%  93%              92%                                   4-chloro-3-ethoxyisocoumarin (5)                                              __________________________________________________________________________     .sup.a Inhibition was performed at 0.1 M Hepes, 0.5 M NaCl, pH 7.5 buffer     10% Me.sub.2 SO and 25° C. Reactivation was carried out in the         presence of 0.4 M of NH.sub.2 OH, and occurred immediately after the          addition of NH.sub.2 OH.                                                      .sup.b Enzyme activity was measured after removal of excess inhibitor by      centrifugation at 0° C. with centricon10 microconcentrator and         staying at 25° C. for two days.                                   

                                      TABLE IV                                    __________________________________________________________________________    Effect of Streptavidin and Avidin on Reactivation of inhibited                Chymotrypsin and PPE.sup.a.                                                                    % of Regained Enzyme Activity                                                 Chymotrypsin     PPE                                                          Buffer                                                                            +Streptavidin                                                                         +Avidin                                                                            Buffer                                                                            +Streptavidin                                                                         +Avidin                         __________________________________________________________________________    7-(6-biotinylaminocaproyl)amino-                                                               15% 39%     12%                                              4-chloro-3-(2-phenylethoxy)IC (3)                                             7-biotinylamino-4-chloro-                                                                      10% 27%     10%                                              3-(2-phenylethoxy)IC (1)                                                      7-(6-biotinylaminocaproyl)amino-  37% 43%     32%                             4-chloro-3-propyloxyIC (4)                                                    7-biotinylamino-4-chloro-         35% 51%     32%                             4-chloro-3-propyloxyIC (2)                                                    __________________________________________________________________________     .sup.a Percentage of regained enzymatic activity was measured in 0.1 M        Hepes, 0.5 M NaCl, pH 7.5 buffer, 8% Me.sub.2 SO and at 25° C.         Excess inhibitor was removed from enzymeinhibitor mixture by                  centrifugation twice with Amicon microconcentrator10. Then 3-4 units of       avidin or strepavidin was added and enzymatic activity was monitored for      two days.                                                                

What is claimed is:
 1. A compound of the formula: ##STR4## or apharmaceutically acceptable salt thereof, wherein Z is selected from thegroup consisting of H, halogen, C₁₋₆ alkyl, C₁₋₆ fluorinated alkyl, C₁₋₆alkyl substituted with R¹, C₁₋₆ fluorinated alkyl substituted with R¹,C₁₋₆ alkoxy, C₁₋₆ fluorinated alkoxy, C₁₋₆ alkoxy substituted with R¹,C₁₋₆ fluorinated alkoxy substituted with R¹, C₁₋₆ alkyl with a phenylgroup attached to the alkyl group, C₁₋₆ alkoxy with a phenyl groupattached to the alkoxy group, C₁₋₆ alkyl with an attached phenyl groupsubstituted with R², C₁₋₆ alkyl with an attached phenyl groupdisubstituted with R², C₁₋₆ alkoxy with an attached phenyl groupsubstituted with R², C₁₋₆ alkoxy with an attached phenyl groupdisubstituted with R²,wherein R² represents halogen, COOH, OH, CN, NO₂,NH₂, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆ alkylamine, C₁₋₆ dialkylamine, C₁₋₆alkyl-O--CO--, C₁₋₆ alkyl-O--CO--NH--, or C₁₋₆ alkyl-S--, wherein R¹represents halogen, COOH, OH, CN, NO₂, NH₂, C₁₋₆ alkoxy, C₁₋₆alkylamine, C₁₋₆ dialkylamine, C₁₋₆ alkyl-O--CO--, C₁₋₆alkyl-O--CO--NH--, C₁₋₆ alkyl-S-, or tosylamino, wherein Spacer is anorganic structure which is 3-24 Å long and including a backbonecomprising at least one of the members of the group consisting of --CH₂--CH₂ --, --CO--NH--, --NH--CO--, --CH₂ --CO--, --CH₂ --NH--, --NH--CH₂--, and --C₆ H₄ --, wherein T represents --NH--, --O--, or --S--, Y isselected from the group consisting of H, halogen trifluoromethyl,methyl, OH and methoxy.
 2. A compound of the formula: ##STR5## or apharmaceutically acceptable salt thereof, wherein Z is selected from thegroup consisting of H, halogen, C₁₋₆ alkyl, C₁₋₆ fluorinated alkyl, C₁₋₆alkyl substituted with R¹, C₁₋₆ fluorinated alkyl substituted with R¹C₁₋₆ alkoxy, C₁₋₆ fluorinated alkoxy, C₁₋₆ alkoxy substituted with R¹,C₁₋₆ fluorinated alkoxy substituted with R¹, C₁₋₆ alkyl with a phenylgroup attached to the alkyl group, C₁₋₆ alkoxy with a phenyl groupattached to the alkoxy group, C₁₋₆ alkyl with an attached phenyl groupsubstituted with R², C₁₋₆ alkyl with an attached phenyl groupdisubstituted with R², C₁₋₆ alkoxy with an attached phenyl groupsubstituted with R², C₁₋₆ alkoxy woith an attached phenyl groupdisubstituted with R²,wherein R² represents halogen, COOH, OH, CN, NO₂,NH₂, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆ alkylamine, C₁₋₆ dialkylamine, C₁₋₆alkyl-O--CO--C₁₋₆ alkyl-O--CO--NH--, or C₁₋₆ alkyl-S--, wherein R¹represents halogen, COOH, OH, CN, NO₂, NH₂, C₁₋₆ alkoxy, C₁₋₆alkylamine, C₁₋₆ dialkylamine, C₁₋₆ alkyl-O--CO--, C₁₋₆alkyl-O--CO--NH--, C₁₋₆ alkyl-S--, or tosylamino, spacer represents--[NH--(CH₂)_(n) --CO]_(n) --, --[NH--(CH₂)_(n) --NH--CO]_(n) --,--(NH--C₆ H₄ --CO)_(n) --, --(NH--C₆ H₄ --NH--CO)_(n) --,--NH--(CH₂)_(n) --CO--NH--(CH₂)_(n) --NH--CO--, --NH--(CH₂)_(n)--CO--NH--(CH₂)₃ --NH--(CH₂)₃ --NH--CO--CH₂ CH₂ --CO--, wherein n=1-6,wherein T represents --NH--, --O--, or --S--, Y is selected from thegroup consisting of H, halogen, trifluoromethyl, methyl, OH and methoxy.3. A compound of the formula: ##STR6## or a pharmaceutically acceptablesalt thereof, wherein Z is selected from the group consisting of H,halogen, C₁₋₆ alkyl, C₁₋₆ fluorinated alkyl, C₁₋₆ alkyl substituted withR¹, C₁₋₆ fluorinated alkyl substituted with R¹, C₁₋₆ alkoxy, C₁₋₆fluorinated alkoxy, C₁₋₆ alkoxy substituted with R¹, C₁₋₆ fluorinatedalkoxy substituted with R¹, C₁₋₆ alkyl with a phenyl group attached tothe alkyl group, C₁₋₆ alkoxy with a phenyl group attached to the alkoxygroup, C₁₋₆ alkyl with an attached phenyl group substituted with R²,C₁₋₆ alkyl with an attached phenyl group disubstituted with R², C₁₋₆alkoxy with an attached phenyl group substituted with R², C₁₋₆ alkoxywoith an attached phenyl group disubstituted with R²,wherein R²represents halogen, COOH, OH, CN, NO₂, NH₂, C₁₋₆ alkyl, C₁₋₆ alkoxy,C₁₋₆ alkylamine, C₁₋₆ dialkylamine, C₁₋₆ alkyl-O--CO--, C₁₋₆alkyl-O--CO--NH--, or C₁₋₆ alkyl-S--, wherein R¹ represents halogen,COOH, OH, CN, NO₂, NH₂, C₁₋₆ alkoxy, C₁₋₆ alkylamine, C₁₋₆ dialkylamine,C₁₋₆ alkyl-O--CO--, C₁₋₆ alkyl-O--CO--NH--, C₁₋₆ alkyl-S--, ortosylamino, wherein R is the side chain of a side chain blocked orunblocked amino acid residue selected from the group consisting ofalanine, valine, leucine, isoleucine, proline, methionine, methioninesulfoxide, phenylalanine, tryptophan, glycine, serine, threonine,cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid,lysine, arginine, histidine, phenylglycine, beta-alanine, norleucine,norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid,citrulline, hydroxyproline, ornithine, homoarginine, sarcosine, indoline2-carboxylic acid, 2-azetidinecarboxylic acid, pipecolinic acid(2-piperidine carboxylic acid), O-methylserine, O-ethylserine,S-methylcysteine, S-ethylcysteine, S-benzylcysteine, NH₂ --CH(CH₂CHEt₂)--COOH, alpha-aminoheptanoic acid, NH₂ --CH(CH₂ -1-naphthyl)-COOH,NH₂ --CH(CH₂ -2-naphthyl)-COOH, NH₂ --CH(CH₂ -cyclohexyl)-COOH, NH₂--CH(CH₂ -cyclopentyl)-COOH, NH₂ --CH(CH₂ -cyclobutyl)-COOH, NH₂--CH(CH₂ -cyclopropyl)-COOH, trifluoroleucine, and hexafluoroleucine,wherein n=1-6, wherein T represents --NH--, --O--, or --S--, Y isselected from the group consisting of H, halogen, trifluoromethyl,methyl, OH and methoxy.
 4. A compound of the formula: ##STR7## or apharmaceutically acceptable salt thereof, wherein Z is selected from thegroup consisting of H, halogen, C₁₋₆ alkyl, C₁₋₆ fluorinated alkyl, C₁₋₆alkyl substituted with R¹, C₁₋₆ fluorinated alkyl substituted with R¹,C₁₋₆ alkoxy, C₁₋₆ fluorinated alkoxy, C₁₋₆ alkoxy substituted with R¹,C₁₋₆ fluorinated alkoxy substituted with R¹, C₁₋₆ alkyl with a phenylgroup attached to the alkyl group, C₁₋₆ alkoxy with a phenyl groupattached to the alkoxy group, C₁₋₆ alkyl with an attached phenyl groupsubstituted with R², C₁₋₆ alkyl with an attached phenyl groupdisubstituted with R², C₁₋₆ alkoxy with an attached phenyl groupsubstituted with R², C₁₋₆ alkoxy woith an attached phenyl groupdisubstituted with R²,wherein R² represents halogen, COOH, OH, CN, NO₂,NH₂, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆ alkylamine, C₁₋₆ dialkylamine, C₁₋₆alkyl-O--CO--, C₁₋₆ alkyl-O--CO--NH--, or C₁₋₆ alkyl-S--, wherein R¹represents halogen, COOH, OH, CN, NO₂, NH₂, C₁₋₆ alkoxy, C₁₋₆alkylamine, C₁₋₆ dialkylamine, C₁₋₆ alkyl-O--CO--, C₁₋₆alkyl-O--CO--NH--, C₁₋₆ alkyl-S--, or tosylamino, AA is a side chainblocked or unblocked amino acid residue, wherein n=1-6, wherein Trepresents --NH--, --O--, or --S--, Y is selected from the groupconsisting of H, halogen, trifluoromethyl, methyl, OH and methoxy.
 5. Acompound of the formula: ##STR8## or a pharmaceutically acceptable saltthereof, wherein Z is selected from the group consisting of H, halogen,C₁₋₆ alkyl, C₁₋₆ fluorinated alkyl, C₁₋₆ alkyl substituted with R¹, C₁₋₆fluorinated alkyl substituted with R¹, C₁₋₆ alkoxy, C₁₋₆ fluorinatedalkoxy, C₁₋₆ alkoxy substituted with R¹, C₁₋₆ fluorinated alkoxysubstituted with R¹, C₁₋₆ alkyl with a phenyl group attached to thealkyl group, C₁₋₆ alkoxy with a phenyl group attached to the alkoxygroup, C₁₋₆ alkyl with an attached phenyl group substituted with R²,C₁₋₆ alkyl with an attached phenyl group disubstituted with R², C₁₋₆alkoxy with an attached phenyl group substituted with R², C₁₋₆ alkoxywith an attached phenyl group substituted with R², C₁₋₆ alkoxy with anattached phenyl group disubstituted with R²,wherein R² representshalogen, COOH, OH, CN, NO₂, NH₂, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆alkylamine, C₁₋₆ dialkylamine, C₁₋₆ alkyl-O--CO--, C₁₋₆alkyl-O--CO--NH--, or C₁₋₆ alkyl-S--, wherein R¹ represents halogen,COOH, OH, CN, NO₂, NH₂, C₁₋₆ alkoxy, C₁₋₆ alkylamine, C₁₋₆ dialkylamine,C₁₋₆ alkyl-O--CO--, or C₁₋₆ alkyl-O--CO--NH--, C₁₋₆ alkyl-S--, ortosylamino, wherein T represents --NH--, --O--, or --S--, Y is selectedfrom the group consisting of H, halogen, trifluoromethyl, methyl, OH andmethoxy.
 6. A compound of the formula: ##STR9## or a pharmaceuticallyacceptable salt thereof, wherein Z is selected from the group consistingof methoxy, ethoxy, propyloxy, or phenylethoxy,wherein Spacer isselected from the group consisting of --[NH--(CH₂)₆ --CO]--,--[NH--(CH₂)₆ --CO]₂ --, or --[NH--(CH₂)₂ --NH--CO--CH₂ --NH--CO]--. 7.A compound of the formula: ##STR10## Z is selected from the groupconsisting of methoxy, ethoxy, propyloxy, or phenylethoxy.